The influence of LPL S447X variants and obesity on lipid profile, oxidative stress, and the risk of T2DM: A case‐control study

Abstract Aims The present study aimed to investigate the association between lipoprotein lipase (LPL) S447X polymorphism and type 2 diabetes mellitus (T2DM), obesity, lipid profile, and oxidative stress parameters in a population from the Kurdistan region of Iraq. Method We studied 250 adults (51% female and 49% male) aged 45–65 years in four groups, obese and normal body mass index (BMI) diabetic patients versus healthy normal BMI and obese individuals as controls. Lipid profile and oxidative stress parameters were analyzed by colorimetric assay. The LPL S447X genotypes were detected by polymerase chain reaction (PCR)‐restriction fragment length polymorphism. Results We found that the obese diabetic group had higher levels of triglycerides (TG), cholesterol, and low‐density lipoprotein‐cholesterol, and lower level of high‐density lipoprotein‐cholesterol than other groups. Obese diabetic patients had higher anthropometric indices than nonobese diabetic patients, obese and normal BMI controls. The levels of TG and total oxidative status (TOS) were significantly lower and higher, respectively, in normal BMI controls than in obese controls. Obese diabetic patients had a lower level of total antioxidant capacity than nondiabetic obese controls. The level of TOS was lower in nondiabetic controls compared to the patient groups. Obese diabetic patients had the highest TOS and malondialdehyde levels. The LPL SX genotype was associated with decreased the risk of T2DM by 79% (odds ratio [OR] = 0.21; 95% confidence interval [CI]: 0.05–0.81, p = 0.03). Also, the presence of this genotype reduced the risk of obesity by 39% (OR = 0.61; 95% CI: 0.07–4.90, p = 0.6). In all individuals, the presence of the SX genotype was associated with significantly lower levels of fasting blood sugar (FBS) and TOS. Conclusion We report the influence of obesity on lipid profile in diabetic and nondiabetic individuals and the effect of LPL SX genotype on decreased risk of T2DM and reduced levels of FBS and TOS.


| INTRODUCTION
Diabetes mellitus (DM), a complex metabolic disorder, over decades has become a significant subject of medical investigation. Type 2 diabetes mellitus (T2DM) along withT1DM are the leading causes of the burden of diabetes worldwide. 1 The aetiological risk factors of T2DM include age, family history, ethnicity, diet quality, and metabolic disorders. 1,2 Dyslipidemia takes the main role in distributed vascular lesions, is more prevalent in diabetic patients, and places them at a remarkably higher risk for cardiovascular disease (CVD). 3,4 Dyslipidemia is characterized by abnormal levels (above the normal range) of one or more lipid profile parameters. 2 Multiple investigations have demonstrated a correlation between dyslipidemia and the quality of T2DM control in patients. 5 A Japanese cohort study on T2DM patients illustrated serum triglycerides (TG) as a much more influential factor than the A1 C hemoglobin for predicting CVD in DM patients, even in those with a higher level of blood glucose. 6 Furthermore, dyslipidemia is associated with oxidative stress.
Oxidative stress-induced lipid peroxidation contributed to endothelial dysfunction, consequently causing the formation of atherosclerotic plaques and the incidence of diabetic nephropathy. 6,7 Lipoprotein lipase (LPL) is a glycoprotein enzyme located on chromosome 8. 8 LPL enzyme has a known vital role in lipid metabolism via producing free fatty acids and monoacyl glycerol for tissue metabolic functions and energy storage by hydrolysis of chylomicrons and TG. 8 Also, LPL possibly has a connection role in the uptake of lipoprotein by the cells and therefore is related to the regulation of plasma lipoproteins levels. 9 More than 40 mutations have been detected in the locus of LPL gene in humans until now. 8 In the LPL gene the C to G substitution (Ser 447 →Stop) produces the LPL protein with C-terminal end truncation by two amino acids. 10 LPL S447X polymorphism has been reported to be involved in the DM pathogenesis. 8,10 Studies have suggested that the LPL S447X polymorphism is associated with raising LPL protein secretion and plasma post-heparin activity and also could decrease the plasma level of TG. 11,12 Since the exact mechanism of lipid metabolism dysfunction in diabetes is not fully understood and also the effect of ethnicity on the identification of genes study should not be ignored, the current study was performed to examine the possible association of LPL S447X variants with T2DM and obesity. Also, the effect of LPL S447X genotypes on the levels of oxidative stress, anthropometric and lipid profile parameters were investigated in a population from the Kurdistan of Iraq.

| Study design and population
The present case-control study was carried out in Sulaimani, the Kurdistan region of Iraq. Data was obtained from the medical and paraclinical information of 250 adults from Sulaimani in the Kurdistan region of Iraq. All the participants were recruited from those who referred to the Sulaimani Endocrine and Diabetic Center, public health laboratory,

| Inclusion and exclusion criteria
Individuals aged 45-65 years with T2DM and nondiabetic people with and without obesity were included in the investigation. People with underlying disorders that may affect the study results, such as T1DM, thyroid, and renal dysfunction, CVDs, and malignancy, and people who received medication for dyslipidemia and also pregnant women, were excluded from the investigation.
Obesity was defined as the body mass index (BMI) above 30 kg/ m 2 . BMI was calculated by dividing weight (kg) by the square of height (m 2 ). Also, all the anthropometric indices such as waist circumference, hip circumference, and wrist circumference were measured by a 0.1 cm accuracy stadiometer.

| Sample collection and measurements
Blood samples were drawn after an overnight fasting. A total of 5 ml of venous blood was obtained from each participant. For DNA extraction, falcon tubes containing ethylenediaminetetraacetic acid were used to store whole blood samples (a volume of 2 ml). In the remaining samples serum was separated for biochemical analysis. FBS was detected using the standard enzymatic method by an automated chemical analyzer (Cobas c311). Lipid profile, including total cholesterol (Chol), TG, highdensity lipoprotein-cholesterol (HDL-C), and low-density lipoproteincholesterol (LDL-C), were measured with an enzymatic colorimetric assay. Total antioxidant capacity (TAC) was analyzed by a colorimetric assay kit (Kiazist Co.). In this method a color solution is produced by a chromogen reagent affected by the reduction of Cu 2+ to Cu + by antioxidants at the wavelength of 450 nm, which was detected via a spectrophotometer. Total oxidative status (TOS) was also measured by a colorimetric method kit (Kiazist Co.). In this method, the ferrous ion (Fe 2+ ) in the presence of oxidants is converted to the ferric ion (Fe 3+ ) that, in the presence of the chromogen, produces a colored solution with an absorbance at the wavelength of 550-580 nm. The malondialdehyde (MDA) concentration was determined colorimetrically using thio-barbituric acid method. 13

| Genotyping
The phenol-chloroform method was used for DNA extraction. 14 The variants of the LPL S447X were detected via polymerase chain reaction (PCR)-restriction fragment length polymorphism. The PCR was performed using 20 pmol of each primer (forward primer: 5′-TACACTAGCAATGTCTAGGTGA-3′ and the reverse primer: 5′-TCAGCTTTAGCCCAGAATGC-3′). The 488-bp PCR product was digested with Mnl1 restriction enzyme as previously described (10).

| Statistical analysis
Data analysis was carried out by SPSS software (SPSS Inc. SPSS for windows, version 16.0.), and the significance level was considered at p < 0.05. In Table 2, which represents the demographic and clinical features, the qualitative variable gender is shown as a number and percentage. The χ 2 test analyzed the difference between the groups based on gender. The quantitative variables are presented as mean and standard division (SD), and the Mann-Whitney U test was used to detect the difference in the investigated variables between groups pairwise, including diabetic patients with and without obesity, controls with and without obesity, obese patients and controls, and nonobese patients and controls. Furthermore, Kruskal-Wallis was performed to examine variables among all four groups. In Table 3, the χ 2 test compared the distribution of the LPL S447X genotypes in patients and controls. Also, association between the LPL S447X variants with the risk of T2DM and obesity was investigated by a multivariable binary logistic regression model, which was adjusted for gender, age, lipid profile, and oxidative stress parameters.

Anthropometric and biochemical parameters comparing all T2DM
patients and all controls are demonstrated in Table 1. Significantly higher levels of waist and wrist circumference, FBS, total cholesterol, LDL-C, TOS, and MDA, and significantly lower levels of HDL-C and TAC were detected in patients compared to controls ( Table 1). As given in Table 2. the mean age of the study population was 53.3 ± 5.3 years, and there was no difference between the study groups according to the age. Based on the χ 2 test, the female population was significantly higher than the male population in the four study groups (p = 0.01). The anthropometric assessment showed a significant difference between all groups, T2DM with and without obesity, T2DM and controls, obese patients and controls. Only normal BMI group of T2DM patients and controls had an insignificant difference regarding anthropometric parameters (Table 2). In addition, the waist-to-hip circumference ratio (WHR) was not significantly different comparing groups. The WHR in both sexes were compared separately. According to the WHO criteria (WHR of 0.90 or higher in men and 0.85 or more in women are considered for defining obesity) 15 both men and women were obese in all studied groups. In males there were WHR of 0.94 ± 0.06, 0.91 ± 0.11, 0.93 ± 0.06, and 0.91 ± 0.05 in obese patients, normal BMI patients, obese controls and normal BMI controls, respectively. In women the WHR levels of 0.91 ± 0.25, 0.88 ± 0.1, 0.85 ± 0.06, and 0.85 ± 0.05 in obese patients, normal BMI patients, obese controls and normal BMI controls, respectively were detected.
ObeseT2DM patients had the highest levels of cholesterol, but in the pairwise examination, the statistically significant difference was found in controls with and without obesity (p = 0.01). Normal BMI diabetic patients had significantly higher LDL-C level (120.7 ± 34.1 mg/dl) than normal BMI controls (108 ± 22.1 mg/dl, p = 0.01). However, the mean HDL-C level was significantly different comparing obese diabetic patients and normal BMI controls, and normal BMI individuals had the highest HDL-C level (50.9 ± 14.3 mg/dl). The TG level was significantly lower in normal BMI individuals than in obese individuals (104.1 ± 42.1 vs. 126.9 ± 44.4 mg/dl, p = 0.004). However, the TOS level was considerably higher in the first group ( Table 2).
The TAC level was compared between obese T2DM patients (3 ± 0.7 nmol/ml) and obese controls (3.3 ± 0.8 nmol/ml) that was significantly different (p = 0.03), and obese diabetic patients had a lower level of TAC than nondiabetic obese controls. The concentration of TOS showed a lower level in nondiabetic controls compared to the patient groups. Furthermore, there was a significant difference between patients with and without obesity. Obese-diabetic patients          in individuals with T2DM. 17 Gavra and colleagues, through the study on T2DM, observed that the carriers of the LPL SX genotype had significantly more negative oral fat tolerance test response (TG postprandial). 18 However, the studies of Evans et al. 19  polymorphism as a cardioprotective phenotype is dependent on its role as a connecting bridge for uptake of the lipoproteins by hepatocyte. 25 In the present study the HDL-C level was significantly higher comparing individuals with WHR < 0.90 than those with WHR ≥ 0.90.
The waist-to-hip circumference ratio assesses distribution or location of fat around the abdomen and hip. The higher value indicates high risk of CVD. It has been reported that this ratio affects the levels of total cholesterol and the ratio of LDL-C to HDL-C. 26

| CONCLUSION
The current study found that the obese diabetic patients had higher levels TG, Chol, LDL-C, and lower level of HDL-C than the control group (obese and normal BMI) and even nonobese diabetic

ACKNOWLEDGMENT
Project number: 4000243. Also, we declared that the source of financing the project was no role or involvement in study design; collection, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication. AHMED ET AL. | 7 of 8

CONFLICTS OF INTEREST
The authors declare no conflicts of interest. Zohreh Rahimi is an Editorial Board member of Health Science Reports. and a co-author of this article. To minimize bias, they were excluded from all editorial decision-making related to the acceptance of this article for publication.

DATA AVAILABILITY STATEMENT
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ETHICS STATEMENT
The research followed the tenets of the Declaration of Helsinki. The Ethics Committee of Kermanshah University of Medical Sciences approved this study (IR.KUMS.REC. 1400.140). All participants entered the study after they were fully informed of the process and signed written consent.

TRANSPARENCY STATEMENT
The lead author Zohreh Rahimi, Maryam Kohsari affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.